U.S. patent application number 10/411977 was filed with the patent office on 2003-10-23 for novel imaging probes.
Invention is credited to Yalpani, Manssur.
Application Number | 20030198599 10/411977 |
Document ID | / |
Family ID | 29250866 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030198599 |
Kind Code |
A1 |
Yalpani, Manssur |
October 23, 2003 |
Novel imaging probes
Abstract
The present invention relates to fluorinated and paramagnetic
polyuronides (Formulas I-IV) and proteins useful as imaging probes,
diagnostic agents and contrast agents. Additionally, the present
invention relates to imaging methods employing the present
compounds of Formulas I-IV.
Inventors: |
Yalpani, Manssur; (Rancho
Sante Fe, CA) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
2421 N.W. 41ST STREET
SUITE A-1
GAINESVILLE
FL
326066669
|
Family ID: |
29250866 |
Appl. No.: |
10/411977 |
Filed: |
April 11, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60372501 |
Apr 11, 2002 |
|
|
|
Current U.S.
Class: |
424/9.35 ;
424/423; 536/123; 536/3 |
Current CPC
Class: |
C08B 37/0084 20130101;
B82Y 5/00 20130101; A61K 49/1866 20130101; A61K 49/126 20130101;
A61K 49/0054 20130101; A61K 49/0093 20130101; A61K 49/0043
20130101; A61K 49/0002 20130101; A61K 49/1863 20130101 |
Class at
Publication: |
424/9.35 ; 536/3;
536/123; 424/423 |
International
Class: |
A61K 049/00; C08B
037/04; C08B 037/00 |
Claims
I claim:
1. A fluorinated or paramagnetic polyuronide polymer comprising a
polymer of Formula I, II , III or IV 19and pharmaceutically
acceptable salts thereof for the polymers that are capable of
forming salts, wherein for Formula I: R.sub.1 represents OH, OX, X;
R.sub.2 represents OH, OX, X; R.sub.3 represents OH, OY, OX, NHX,
alkyl, alkoxyalkyl; R.sub.4 represents C.dbd.O, CH.sub.2, CNX,
CF.sub.2; for Formula II: R.sub.1 represents OH, OX, X, OR.sub.4;
R.sub.2 represents OH, OX, X, OR4; R.sub.3 represents OH, Y, X;
R.sub.4=acyl, alkly or aryl for Formula III: R.sub.1 represents OH,
OX, X; R.sub.2 represents OH, OX, X; R.sub.3 represents H,
N(X).sub.3; for Formula IV: R.sub.1 represents OH, OX, X; R.sub.2
represents OH, OX, X, --OA(X)O-- or OA(R.sub.5)O--; R.sub.5
represents alkyl or acyl and A represents a non-paramagnetic ion
wherein M is any paramagnetic ion of the transition metal or
lanthanide series, and "a" is a whole number and "b" is 1/a;
wherein for all of the formulas I-IV X represents a fluorine
containing moiety, a luminescent residue, a fluorescent residue, a
fluorinated luminescent residue or a fluorinated fluorescent
residue; Y represents CH.sub.2C(OH)CH.sub.3; m, p, x, y, z
represents 0-150; and n is from 10-10,000 inclusive.
2. The polyuronide polymer, according to claim 1, comprising a
compound selected from the group consisting of a.
heptafluorobutyryl alginic acid of the formula 20wherein m+n=1 b.
6-[3-[2-(perfluorohexyl)-2-ethoxy]-2-h- ydroxypropyl] alginic acid
of the formula 21wherein m+n=1 c. perfluoro tri-n-butylamine
alginate of the formula 22wherein m+n=1 d.
polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.-methyl-car-
boxylate-.omega.-carboxylate propylene glycol alginate of the
formula 23wherein m+n=1 e. 6-[2-(Perfluorohexyl)-2-hydroxy] DANSYL
alginic acid of the formula 24wherein m+n=1 f.
3-[(hexafluoropropyl)-2-hydroxy] alginic acid of the formula
25wherein m+n=1 g. perfluorophenylhydrazone alginic acid of the
formula 26wherein m+n=1 h. polytetrafluoroethyleneox-
ide-co-difluoromethyleneoxide-.alpha.-difluoroacetic
acid-.omega.-difluoroacetyl fluorescein alginate of the formula
27wherein m+n=1 i.
Polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-
-.alpha.-difluoroacetic acid-.omega.-difluoroacetyl propylene
glycol alginate of the formula 28wherein m+n=1 j.
perfluorobenzamide alginate of the formula 29wherein m+n=1 k.
perfluoroaniline alginate of the formula 30wherein m+n=1 l.
3-[(hexafluoropropyl)-2-hydroxy] propyleneglycol alginate of the
formula 31wherein m+n=1 m.
polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.-tolylureth-
ane-.omega.-tolylisocyanate propylene glycol alginate of the
formula 32wherein m+n=1 n. perfluoro-3,6,9-trioxatridecanoate
alginate of the formula 33wherein m+n=1 o.
3,5,5"-tris(trifluoromethyl)octafluorohexanoa- te alginate of the
formula 34wherein m+n=1 p. 3,5,5'-tris(trifluoromethyl-
)octafluorohexanoate propylene glycol alginate of the formula
35wherein m+n=1 q. methyl perfluorohexadecanoate alginate of the
formula 36wherein m+n=1 r. heptafluorobutyryl hydroxyethyl starch,
and s. perfluorotri-n-butylamine alginate
3. The fluorinated and/or paramagnetic polymer according to claim
1, wherein M is selected from the group consisting of gadolinium
(III), iron (III), manganese (II and II), chromium (III), copper
(II), dysprosium (III), terbium (III), holmium (III), erbium (III),
and europium (III).
4. The fluorinated and/or paramagnetic polymer according to claim
1, wherein a. In Formula I R.sub.1 is OH or OX, R.sub.2 is OH or
OX, R.sub.3 is OH, and R.sub.4 is C.dbd.O; b. In Formula II R.sub.1
is OH or OX, R.sub.2 is OH or OX, R.sub.3 is OH, and R.sub.4 is
C.dbd.O; c. In Formula III R.sub.1 is OH, R.sub.2 is OH, and
R.sub.3 is H, N(X).sub.3 and d. In Formula IV R.sub.1 is OH or
--OA(X)O--, R.sub.2 is OH or --OA(X)O--, and M is gadolinium (III),
iron (III), manganese (II) or dysprosium (III).
5. The fluorinated and/or paramagnetic polymer according to claim
1, wherein X is fluoroalkyl, fluoroaryl, fluoroacyl,
perfluoroalkyl, perfluoroaryl, perfluoroacyl, perfluoropolymer,
fluoroamine, fluorocarbamate, fluorotriazine, fluorosulfonylalkyl
derivatives, F, CF.sub.3, COC.sub.xF.sub.y, CF.sub.3CO.sub.2,
C.sub.xF.sub.yH.sub.z,
([CH.sub.2].sub.mO).sub.x(CH.sub.2CF.sub.2O).sub.y(CF.sub.2CF.sub.2O).sub-
.z(CF.sub.2).sub.2CF.sub.2CH.sub.2O(CH.sub.2).sub.pOH,
CH.sub.2C(OH)C.sub.xF.sub.yH.sub.z, C.sub.xF.sub.yH.sub.zO.sub.p,
COC.sub.xF.sub.yH.sub.z,
OCH.sub.2C.sub.xF.sub.z[C.sub.xF.sub.zO].sub.mF,
CH.sub.2C(CH.sub.3)CO.sub.2C.sub.xH.sub.z(CF.sub.2).sub.mCF.sub.3,
CH.sub.2(CF.sub.2O).sub.x(CF.sub.2CF.sub.2O).sub.y(CF.sub.2O).sub.zCF.sub-
.2CH.sub.2OH, NHC.sub.xF.sub.yH.sub.zO.sub.p,
CH.sub.2CF.sub.2O[CF.sub.2CF-
.sub.2O].sub.mCF.sub.2OCF.sub.2CH.sub.2OH,
COC.sub.xH.sub.z(CF.sub.2).sub.- mCF.sub.3,
CO--CF.sub.2O[CF.sub.2CF.sub.2O].sub.nCF.sub.2OCF.sub.2CO.sub.2- H,
CO--CF(CF.sub.3)[CF(CF.sub.3)CF.sub.2O].sub.mF
([CH.sub.2].sub.mO).sub.-
x(CH.sub.2CF.sub.2O).sub.y(CF.sub.2CF.sub.2O).sub.zCF.sub.2CH.sub.2O(CH.su-
b.2).sub.pOH, SO.sub.2[CF.sub.2].sub.xCF.sub.3, CF.sub.3SO.sub.3,
N[C.sub.xF.sub.yH.sub.z].sub.p,
C.sub.xH.sub.zCO.sub.2C.sub.xH.sub.z(CF.s- ub.2).sub.mCF.sub.3, or
COC.sub.xF.sub.y[C.sub.pF.sub.zO].sub.mF,; Y represents
CH.sub.2C(OH)CH.sub.3; m, p, x, y, z represents 0-150 and n is from
10-10,000 inclusive.
6. A paramagnetic alginate bead comprising paramagnetic gadolinium
alginate beads or paramagnetic iron oxide alginate beads.
7. A paramagnetic particle comprising a paramagnetic iron oxide
alginate-coated particle, a paramagnetic Iron oxide alginate-coated
particle or a paramagnetic iron oxide dextran-coated particle.
8. A bioactive protein paramagnetic conjugate comprising a
bioactive protein conjugated or complexed with a paramagnetic ion
of the transition metal or lanthanide series.
9. The bioactive protein paramagnetic conjugate according to claim
8, wherein the paramagnetic protein conjugate is iron oxide annexin
conjugate, a superparamagnetic iron oxide annexin conjugate or a
fluorescent, superparamagnetic iron oxide annexin conjugate.
10. The fluorinated polyuronide polymer according to claim 1,
wherein the polyuronide is an alginate and the alginate has a
.alpha.-L-guluronic acid content of at least 50% by weight.
11. An implant for a mammalian host which comprises: a. live cells
that produce a desired biologically active compound and b. one or
more polymers of claim 1 wherein the polymer(s) encapsulate the
live cells and inhibit host immune reactions to the implant and
allow evaluation of the implant by MRI examination.
12. The implant according to claim 11, wherein the polymer is a
fluorinated or paramagnetic alginate.
13. The implant according to claim 11, wherein the cell is an
insulin producing human or porcine pancreatic B islet cell.
14. The implant according to claim 11, wherein the polymer is a
fluorinated or paramagnetic alginate.
15. A method of improving the effectiveness of magnetic resonance
imaging (MRI) which comprises: a. administering an effective amount
of one or more fluorinated and/or paramagnetic polymers of claim 1
to a patient; b. subjecting the patient to an MRI of a tissue/organ
where the administered polymer is expected to accumulate; and c.
evaluating the tissue/organ from the MRI images obtained.
16. The method according to claim 15 wherein the polymer is a
fluorinated polyuronide.
17. The method according to claim 15, wherein the polymer is a
paramagnetic polyuronide.
Description
CROSS-REFERENCE TO A RELATED APPLICATION
[0001] This application claims the benefit of provisional patent
application Serial No. 60/372,501, filed Apr. 11, 2002, which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel imaging probes and
methods for using the probes in diagnostic imaging processes and
other imaging processes to determine physiological functions.
Additionally, the present invention relates to implants
encapsulated with the present imaging probes.
BACKGROUND OF THE INVENTION
[0003] Diabetes is a devastating disease of immense proportions. It
is characterized by an impaired glucose metabolism that leads to,
among other things an elevated blood glucose level (hyperglycemia)
in diabetic patients. Type 1 diabetes is caused by autoimmune
destruction of insulin-secreting .beta.-cells within islets of
Langerhans in the pancreas. Diabetes is classified into type 1, or
insulin dependent diabetes mellitus (IDDM), which arises when a
patient's .beta.-cells cease producing insulin in their pancreatic
glands, and type 2, or non-insulin dependent diabetes mellitus
(NIDDM), which occurs in patients with an impaired insulin
metabolism and .beta.-cell malfunction. NIDDM usually takes decades
to develop and is characterized sequentially by hyperinsulinemia,
elevated triglycerides, high blood glucose and finally in late
stages .beta. cell fatigue, where insulin levels drop precipitously
usually requiring insulin administration to the patient. In IDDM
patients, the .beta.-cells are selectively destroyed by an
autoimmune process that involves lymphocyte infiltration. Early in
the course of NIDDM, .beta.-cell mass increases to meet the demand
for more insulin. Loss of .beta.-cell mass may then occur as NIDDM
advances. .beta.-Cells secrete insulin in response to changes in
blood glucose concentration in highly regulated fashion and are
responsible for achieving minute-to-minute regulation within
physiological levels. Insulin deficiency results in prolonged
hyperglycemia with serious long-term complications. Current
treatments (e.g. insulin injections) do not provide tight
regulation of blood glucose levels and thus do not alleviate the
long-term complications of diabetes. Both naked and encapsulated
islet transplantation are being explored as alternative treatments
that can provide more physiological blood glucose level control.
Islet transplantation is a promising method for restoring
normoglycemia and alleviating the long-term complications of
diabetes. Widespread application of islet transplantation is
hindered by the limited supply of human islets and will require a
large increase in the availability of suitable insulin secreting
tissue as well as robust quality assessment methodologies that will
ensure safety and in vivo efficacy.
[0004] The transplantation of immunoprotected insulin-secreting,
glucose-responsive cells is a promising method for the long-term
treatment of type 1 diabetes. A limitation that needs to be
addressed before this methodology is implemented at a large-scale
is cell availability. Human islets cannot be amplified in culture
while retaining their differentiated secretory properties, so
tissue from at least one human donor is needed for a single
treatment of one recipient. The tissue availability limitation can
be addressed by employing xenogeneic tissue (such as porcine
islets) that is protected from the host's immune response.
Immunoprotection can be achieved by enclosing the cells in a
permselective membrane allowing passage of low molecular weight
nutrients and metabolites, including insulin, but excluding larger
antibodies and cytotoxic cells of the host. Most of the
experimental work on encapsulated cell therapies has employed
alginate as the encapsulating matrix. This methodology is
particularly promising because it has the potential of restoring
physiological regulation of blood glucose levels without the need
for life long immunosupressive therapy. The feasibility of this
approach in restoring normoglycemia has been demonstrated for
diabetic animals and human with promising results (P. Soon-Shiong,
et al., Lancet, 343, 950-1, 1994; R. P. Lanza, D. M. Kuhtreiber, et
al., Transplant. Res., 28, 820, 1996; E. H. Liu, K. C. Herold,
Trends Endocrinol. Metab. 11, 379-82, 2000; A. M. Shapiro, J. R.
Lakey, et al., N. Engl. J. Med., 343, 230-238, 2000.).
[0005] Individuals at risk for developing IDDM can be identified by
certain techniques. Those at risk for NIDDM are identifiable
through family history and measurement of insulin resistance.
However, little is known about the natural history of .beta.-cell
mass, turnover and cell lifetime, or the course of inflammation in
diabetes. This is attributable to the highly heterogeneous nature
of the pancreas, difficulties in its biopsy, and the low volume of
.beta.-cell mass (only 1-2% of the organ). Although insulin
secretory capacity can be measured, it poorly reflects .beta.-cell
mass. There is therefore a substantial need for diagnostic methods
that would enable (i) high-risk individuals to be monitored prior
to onset of diabetes; (ii) diabetes patients to be monitored over
the course of their disease to determine the exact stage of their
disease; and (iii) also monitoring responses to therapy.
[0006] Current therapeutics for Type 1 diabetics are insulin or
insulin mimetics, while most type 2 diabetic patients are treated
either with agents that stimulate .beta.-cell function or enhance
the patient's tissue sensitivity towards insulin. Several classes
of drugs are available for diabetes therapy. These include:
insulin, or insulin mimetics; insulin sensitizers including (a)
biguanides such as Metformin (b) retinoid-X-receptor (RXR) and
peroxisome proliferator activated receptor (PPAR) agonists, such as
the Thiazolidinedione (glitazone)and PPAR-.gamma. agonists, e.g.,
Rosiglitazone and Troglitazone; (c) sulfonylureas (SU), such as
Gliclazide, Glimepiride, Glipizide, Glyburide, Tolbutamide and
Tolcyclamide; (d) amino acid and benzoic acid derivatives, such as
Nateglinide and Repaglinide; (e) .alpha.-glucosidase inhibitors,
such as Acarbose; (f) cholesterol lowering agents, such as (i)
HMG-CoA reductase inhibitors, e.g., Lovastatin, and other statins),
(ii) bile acid sequestrants, e.g., Cholestyramine (iii) nicotinic
acid (iv) proliferator-activator receptor .alpha.-agonists, such as
Benzafibrate, and Gemfibrozil, (v) cholesterol absorption
inhibitors, e.g., .beta.-sitosterol and (vi) acyl
CoenzymeA:cholesterol acyltransferase inhibitors, e.g., Melinamide,
and (g) Probucol.
[0007] Whilst continuous efforts are directed at developing new
anti-diabetic agents, there is also a considerable need for the
development of materials related to known therapeutic agents that
may display improved bioavailability, functionality or reduced
levels of undesirable effects. There is also a need for new
diagnostic agents that can facilitate elucidation of the mechanism
of insulin release or sensitization and the binding mechanism of
the known anti-diabetic agents to their respective molecular
receptors.
[0008] Fluorocarbon compounds and their formulations have numerous
applications in medicine as therapeutic and diagnostic agents and
as blood substitutes. Fluorine features a van der Waals radius
(1.2A) similar to hydrogen (1.35A). Hydrogen replacement (with F)
does therefore not cause significant conformational changes and
fluorination can lead to increased lipophilicity, enhancing the
bioavailability of many drugs. Fluorinated materials are often
biologically inert and are generally expected to reduce side-effect
profiles of drugs. The carbon-fluorine bond strength (460 kJ/mol in
CH.sub.3F) exceeds that of equivalent C--H bonds. Perfluorocarbons
(PFCs) display high chemical and biological inertness and a
capacity to dissolve considerable amounts of gases, particularly
oxygen, carbon dioxide and air per unit volume. PFCs can dissolve
about a 50% volume of oxygen at 37.degree. C. under a pure oxygen
atmosphere. Fluorocarbon compositions can be used for wound
treatment, as described in U.S. Pat. No. 4,366,169. Fluorocarbon
formulations are also useful in diagnostic procedures, for example
as contrast agents (Riess, J. G., Hemocompatible Materials and
Devices: Prospectives Towards the 21st Century, Technomics Publ.
Co, Lancaster, Pa. USA, Chap 14 (1991); Vox Sanguinis, 61:225-239,
1991).
[0009] Nuclear magnetic resonance (NMR) techniques permit the
assessment of biochemical, functional, and physiological
information from patients. Magnetic resonance imaging (MRI) of
tissue water can be used to measure perfusion and diffusion with
submillimeter resolution. Magnetic resonance spectroscopy may be
applied to the assessment of tissue metabolites that contain
protons, phosphorus, fluorine, or other nuclei. The combination of
imaging and spectroscopy technologies has lead to spectroscopic
imaging techniques that are capable of mapping proton metabolites
at resolutions as small as 0.25 cm.sup.3 (Zakian K L; Koutcher J A;
Ballon D; Hricak H; Ling C C, Semin Radiat Oncol.; 11(1):3-15,
2001). In magnetic resonance angiography (MRA) contrast agents are
used to image the arteries and veins for diagnosing cardiovascular
disease and associated disorders.
[0010] Of particular interest is fluorine's diagnostic value in
non-invasive imaging applications. Apolar oxygen imparts
paramagnetic relaxation effects on .sup.19F nuclei associated with
spin-lattice relaxation rates (R.sub.1) and chemical shifts. This
effect is proportional to the partial pressure of O.sub.2
(pO.sub.2). .sup.19F NMR can therefore probe the oxygen environment
of specific fluorinated species in cells and other biological
structures.
[0011] Noth et al. (Noth U; Grohn P; Jork A; Zimmermann U; Haase A;
Lutz, J., .sup.19F-MRI in vivo determination of the partial oxygen
pressure in perfluorocarbon-loaded alginate capsules implanted into
the peritoneal cavity and different tissues, Magn. Reson. Med.
42(6):1039-47, 1999) employed perfluorocarbon-loaded alginate
capsules in MRI experiments to assess the viability and metabolic
activity of the encapsulated materials. Quantitative .sup.19F-MRI
was performed on perfluorocarbon-loaded alginate capsules implanted
into rats, in order to determine in vivo the pO.sub.2 inside the
capsules at these implantation sites. Fraker et al. reported
recently a related method with perfluorotributylamine (C. Fraker,
L. Invaeradi, M. Mares-Guia, C. Ricordi, PCT WO 00/40252,
2000).
[0012] Ideally, PFC imaging agents should combine the following
features: non-toxic, biocompatible, chemically pure and stable, low
vapor pressure, high fluorine content, reasonable cost and
commercial availability. Additionally, they should meet several
.sup.19F-NMR criteria, including a maximum number of chemically
equivalent fluorines resonating at one or only few frequencies,
preferably from trifluoromethyl functions. Some of the other
spectral criteria have been discussed in detail elsewhere (C. H.
Sotak, P. S. Hees, H. N. Huang, M. H. Hung, C. G. Krespan, S.
Raynolds, Magn. Reson. Med., 29, 188-195, 1993.). For MRI, it would
furthermore be desirable to have control over the amount of
magnetically responsive material for specific uses, and to employ
temperature-responsive and pH-dependent imaging agents for special
uses. These could have applications in MRI-based temperature
monitoring for use in general hyperthermia treatment (see, e.g., S.
L. Fossheim; K. A. ll'yasov, J. Hennig, A. Bjornerud, Acad.
Radiol., 7(12),1107-15, 2000.) of tumors and for monitoring the
efficacy of chemotherapy, respectively (see, e.g., N. Rhagunand, R.
Martinez-Zagulan, S. H. Wright, R. J. Gilles, Biochem. Pharmacol.,
57, 1047-1058, 1999; I. F Tannock, D. Rotin, Cancer Res., 49,
4373-4383, 1989.). Furthermore, water solubility would enhance the
PFC functionality in many biomedical settings, as it would obviate
the need for emulsifiers.
[0013] Although selected efforts have been directed at developing
new fluorinated MRI probes, none are water soluble compounds [e.g.,
perfluoro-[15]-crown-5 ether)], and some are commercially
unavailable [e.g.,
perfluoro-2,2,2',2'-tetramethyl-4,4'-bis(1,3-dioxalane)]. It
appears no attempts have so far focused on screening available PFCs
from the thousands of commercial fluorinated products in order to
identify potentially more suitable MRI probes for biomedical uses.
It seems furthermore that no studies have attempted to establish
structure activity relations (SARs) of related PFCs for MRI
purposes. Noteworthy is also the fact that all PFCs examined to
date have molecular weights under 1,000, typically between 400-600
Da. This is partly a reflection of the specific requirements for
blood substitution agents, but also due to the widely held belief
that higher molecular weight or polymeric fluorinated agents would
not be detectable by .sup.19F-NMR due to anticipated excessive line
broadening, and would therefore be unsuitable. Thus, with the
exception of the polymer-encapsulated PFCs noted above, this
important class of materials had so far been excluded from
consideration.
[0014] Paramagnetic ions, such as gadolinium (Gd.sup.3+) decrease
the T.sub.1. of water protons in their vicinity, thereby providing
enhanced contrast. Gadolinium's long electron relaxation time and
high magnetic moment make it a highly efficient T.sub.1 perturbant.
Since uncomplexed gadolinium is very toxic, gadolinium chelate
probes, such as gadolinium diethylenetriamine pentaacetic acid
(GdDTPA M.sub.w 570 Da), albumin-GdDTPA (Gadomer-17, M.sub.w 35 or
65 kDa), have been employed extensively in MRI of tumors and other
diseased organs and tissues. Several other developmental chelators
have also been reported, including dual-labeled agents,
oligonucleotide-derived, dextran-derived GdDTPA, and TAT and other
peptide-derived chelators. However, presently approved MRI contrast
agents are either not tissue specific, e.g., GdDTPA, or target only
normal tissue, which limits their utility in diagnosis of
metastases or neoplasia. MRI studies with GdDTPA, for instance, do
not correlate with the angiogenic factor or the vascular
endothelial growth factor (VEGF). Attempts have also been made to
overcome the low relaxivities of small Gd-DTPA chelates by
preparing polymer conjugates of Gd(DTPA).sup.(2-) [see e.g., MRA.
Duarte M. G.; Gil M. H.; Peters J. A.; Colet J. M.; Elst L. Vander;
Muller R. N.; Geraldes C. F. G. C., Bioconjug. Chem., 21, 170-177,
2001.]. However, the relaxivity of these polymer conjugates was
only slightly improved and they were also cleared very quickly from
the blood of rats, indicating that they are of limited value as
blood pool contrast agents for MRI.
[0015] Annexin V is a human protein (Mw 36,000) with high affinity
for cells or platelet membranes that, following apoptosis
(programmed cell death), have redistributed phosphatidylserine (PS)
functions from internal to external membrane surfaces (see e.g.,
Verhoven et al. [B. Verhoven, R. A. Schlege, P. Willamson, J. Exp.
Med., 182, 1597-1601, 1995] and Tait et al. [J. F. Tait, D. Gibson,
J. Lab. Clin. Med., 123, 741-748, 1994.]). Apoptosis is an integral
part of the aging and development of the central nervous system
(CNS) and is linked to the pathogenesis of autoimmune and
neurodegenerative diseases, cerebral and micordial ischemia,
vasogenic edema, viral infections, inflammatory demyelinating
diseases, organ and bone marrow transplant rejection, tumor
response to chemotherapy and radiotherapy, and trauma [see e.g., H.
Steller, Science, 267, 1445-1449, 1995; S. M. de la Monte, Y. K.
Sohn, N. Ganju, J. R. Wands, Lab Invest., 158, 1001-1009, 1998.].
Among the neurodegenerative diseases linked to apoptotic events are
Alzheimer's disease, Pick's disease, Parkinson's Disease,
progressive supranuclear palsy, amyotrophic lateral sclerosis, and
diffuse Lewy Body disease. These diseases are believed to share
common neurodegenerative mechanisms, but maintain distinct clinical
and pathological profiles due to atrophy and cell loss in specific
regions of the CNS.
[0016] In view of the ubiquitous role of apoptosis in a broad range
of disorders, a probe that could identify and quantify cell death
in vivo would be of substantial benefit. The study of CNS neuron
apoptosis could be a valuable tool for screening more effective
drugs in the treatment of dementia associated with Alzheimer's and
other diseases. One of the past hurdles in the development of
potential therapies has been the general lack of relevant in vitro
and in vivo diagnostic methodologies to assess the potential of new
therapeutic compounds. Annexin's affinity for PS has been exploited
to study in animals and humans hepatic apoptosis, chemotherapy,
allograft rejection, and thrombosis, using radioisotope-labeled
annexin [see e.g., Blankenberg et al., Proc. Natl. Acad. Sci. USA,
95, 6349-6354, 1998; Ohtsuki K; Akashi K; Aoka Y; Blankenberg F.
G.; Kopiwoda S; Tait J. F.; Strauss H. W. Eur. J. Nucl. Med., 26,
1251-8, 1999. Blankenberg F. G.; Katsikis P. D.; Tait J. F.; Davis
R. E.; Naumovski L.; Ohtsuki K.; Kopiwoda S.; Abrams M. J.; Strauss
H. W., J. Nucl. Med., 40, 184-91, 1999. J. R. Stratton, et al.,
Circulation, 92, 3113-3121, 1995.]. Another PS binding protein, the
C.sub.2 domain of synaptotagmin I, was conjugated to
superparamagnetic iron oxide (SPIO) nanoparticles and used in MRI
to detect apoptotic cells. Zhou, et al, Nature Medicine, Vol. 7,
No.: 11, November 2001.
[0017] Whilst much can be achieved with currently available imaging
and contrast agents, there are still unmet needs for novel
diagnostic agents, particularly for those exploiting biological
specificity. Imaging agents suitable for targeting metastases or
neoplasia would substantially enhance the MRI sensitivity and
utility for tumor detection and prevention. Similarly, imaging
agents suitable for targeting receptors involved in insulin
production and utilization would substantially enhance our
understanding of the diabetes disease process and the function of
anti-diabetic drugs. Although selected efforts have been directed
at developing such new probes, a broader investigation of these
agents is urgently needed. Similarly, new imaging probes are needed
as noninvasive means to detect and image cells, tissues and organs
undergoing apoptosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-C shows spectra of heptafluorobutyryl alginate of
Example 1 in D.sub.2O (a); its expanded trifluoromethyl resonance
(b); and (c) Ca.sup.2+ beads of heptafluorobutyryl alginate in
D.sub.2O (376 MHz .sup.19F-NMR (0.4%)).
[0019] FIGS. 2A-C shows NMR spectra of selected perfluoroalginates:
(a) perfluoropolymer alginate derivative of Example 9; (b)
perfluorophenylhydrazone alginate derivative of Example 7; and (c)
perfluoropolymer alginate derivative of Example 4 (376 MHz
.sup.19F-NMRs (in D.sub.2O)).
BRIEF SUMMARY OF THE INVENTION
[0020] The present invention relates to fluorinated and/or
paramagnetic polyuronides (Formulas I-IV) and fluorinated and/or
paramagnetic proteins useful as imaging probes, diagnostic agents
and contrast agents. Additionally, the present invention relates to
imaging methods employing the present compounds of Formulas I-IV
and the fluorinated/paramagnetic proteins described herein.
[0021] The fluorinated and chemically modified polyuronides of the
present invention include compounds of general formulas I to IV
below: 1
[0022] Where
[0023] For Formula I:
[0024] R.sub.1=OH, OX, X; R.sub.2=OH, OX, X, NHCOX; R.sub.3=OH, OY,
OX, NHX, alkyl, alkoxyalkyl; R.sub.4 represents C.dbd.O, CH.sub.2,
CNX, CF.sub.2; preferably R.sub.1 is OH or OX, R.sub.2 is OH or OX
wherein it is especially preferred that one of R.sub.1 and R.sub.2
is OH while the other is OX
[0025] For Formula II:
[0026] R.sub.1=OH, OX, X, OR.sub.4; R.sub.2=OH, OX, X, OR.sub.4;
R.sub.3=OH, Y ,X, R.sub.4; R.sub.4=acyl, alkyl or aryl; preferably
R.sub.1 is OH or OX, R.sub.2 is OH or OX, R.sub.3 is OH, R.sub.4 is
C.dbd.O, wherein it is especially preferred that one of R.sub.1 and
R.sub.2 is OH while the other is OX
[0027] For Formula III:
[0028] R.sub.1=OH, OX, X; R.sub.2=OH, OX, X; R.sub.3=H, N(X).sub.3
and preferably R.sub.1 is OH, R.sub.2 is OH and R.sub.3 is H or
N(X).sub.3
[0029] For Formula IV:
[0030] R.sub.1=OH, OX, X; R.sub.2=OH, OX, X, --O--A(X)--O-- or
--O--A (R.sub.3)--O--; R3=alkyl, acyl or
[0031] M is any paramagnetic ion of the transition metal or
lanthanide series, including gadolinium (III), iron (III),
manganese (II and III), chromium (III), copper (II), dysprosium
(III), terbium (III), holmium (III), erbium (III), and europium
(III); most preferred are gadolinium (III), dysprosium (III), iron
(III), and manganese (II), and for the ion valency of a=1, b=1;
a=2, b=1/2; a=3, b=1/3; a=4, b=1/4; etc.;
[0032] A is any non-paramagnetic ion, including fluorinated
ammonium salts, e.g., ammonium heptafluorotantatalate (V), ammonium
hexafluorogermanate, ammonium hexafluoroniobate, ammonium
hexafluorophosphate, ammonium hexafluorostannate, ammonium
tetrafluoroborate, antimony(III) or (V) fluoride, barium fluoride,
fluoroboron salts, including boron trifluoride and its derivatives,
fluorolithiates, including lithium tetrafluoroborate and lithium
fluoride, iron (II) fluoride, magnesium fluoride, potassium
fluoride, sodium fluoride, and tetralkylammonium fluoride salts,
including tetrabutylammonium tetrafluoroborate;
[0033] Wherein
[0034] X is a fluorine containing moiety. Suitable fluorine
moieties include fluoroalkyl, fluoroaryl, fluoroacyl,
perfluoroalkyl, perfluoroaryl, perfluoroacyl, perfluoropolymer,
fluoroamine, fluorocarbamate, fluorotriazine, fluorosulfonylalkyl
derivatives, F, CF.sub.3, COC.sub.xF.sub.y, CF.sub.3CO.sub.2,
C.sub.xF.sub.yH.sub.z,
([CH.sub.2].sub.mO).sub.x(CH.sub.2CF.sub.2O).sub.y(CF.sub.2CF.sub.2O).sub-
.z(CF.sub.2).sub.2CF.sub.2CH.sub.2O(CH.sub.2).sub.pOH,
CH.sub.2C(OH)C.sub.xF.sub.yH.sub.z, C.sub.xF.sub.yH.sub.zO.sub.p,
COC.sub.xF.sub.yH.sub.z,
OCH.sub.2C.sub.xF.sub.z[C.sub.xF.sub.zO].sub.mF,
CH.sub.2C(CH.sub.3)CO.sub.2C.sub.xH.sub.z(CF.sub.2).sub.mCF.sub.3,
CH.sub.2(CF.sub.2O).sub.x(CF.sub.2CF.sub.2O).sub.y(CF.sub.2O).sub.zCF.sub-
.2CH.sub.2OH, NHC.sub.xF.sub.yH.sub.zO.sub.p,
CH.sub.2CF.sub.2O[CF.sub.2CF-
.sub.2O].sub.mCF.sub.2OCF.sub.2CH.sub.2OH,
COC.sub.xH.sub.z(CF.sub.2).sub.- mCF.sub.3,
CO--CF.sub.2O[CF.sub.2CF.sub.2O].sub.nCF.sub.2OCF.sub.2CO.sub.2- H,
CO--CF(CF.sub.3)[CF(CF.sub.3)CF.sub.2O].sub.mF
([CH.sub.2].sub.mO).sub.-
x(CH.sub.2CF.sub.2O).sub.y(CF.sub.2CF.sub.2O).sub.zCF.sub.2CH.sub.2O(CH.su-
b.2).sub.pOH, SO.sub.2[CF.sub.2].sub.xCF.sub.3, CF.sub.3SO.sub.3,
N[C.sub.xF.sub.yH.sub.z].sub.p,
C.sub.xH.sub.zCO.sub.2C.sub.xH.sub.z(CF.s- ub.2).sub.mCF.sub.3,
COC.sub.xF.sub.y[C.sub.pF.sub.zO].sub.mF, a luminescent residue, a
fluorescent residue, a fluorinated luminescent residue or a
fluorinated fluorescent residue, Y=CH.sub.2C(OH)CH.sub.3, and m, p,
x, y, z=0-150 and where m is more preferably 10-100, and most
preferably 10-50, and where x, p, y, z are more preferably 10-75,
even more preferably 10-50, and most preferably 10-20. and where n
is more preferably 10-10,000, even more preferably 10-1,000, and
most preferably 10-250. Preferred compounds of Formula IV are those
where R.sub.1 and R.sub.2 are both OH.
[0035] Acyl and alkyl residues of Formulas I to IV comprise
lipophilic moieties, including saturated and unsaturated aliphatic
residues with C.sub.k chains, where k is 2 to 100, more preferably
2-50, and most preferably 2-20, and where aryl residues comprise
aromatic moieties, including benzyl, biphenyl, phenyl, polycyclic
aromatics, and heteroatom-containing aromatics. The novel
fluorinated and paramagnetic polyuronides of Formulas I to IV
comprise polyuronide analogs, wherein said polyuronides are
selected from the group consisting of acacia, alginate, gellan,
glycosaminoglycans, hyaluronate, polymannuronic acid, polyguluronic
acid, pectins, propyleneglycol alginate, acacia, carboxyalkyl
glycans, including carboxymethyl amylose, carboxymethyl cellulose,
carboxymethyl dextran, carboxymethyl starch, starch, hydroxyethyl
starch, hetastarch, pentastarch, dextran, tragacanth and xanthan.
Pharmaceutically acceptable salts of the above fluorinated
polyuronides are also contemplated by the present invention. When
more than one designated substituent or moiety (for example X)
appears in a formula for a compound, then the substituent can be
the same or different at the various positions of that substituent
in the formula for that compound.
[0036] Disclosed are novel compositions comprising fluorinated and
chemically modified proteins with activity or affinity for cells,
cell surfaces, membranes, cell surface receptors, and receptors
regulating biological membrane channels. The fluorinated and/or
paramagnetic proteins are useful as imaging probes and diagnostic
agents. Exemplary proteins include fluorinated analogs of Annexin V
and synaptotagmin I and superparamagnetic Annexin V iron oxide
conjugates and synaptotagmin I iron oxide conjugates.
DETAILED DESCRIPTION OF THE INVENTION
[0037] In practicing the present invention a polyuronide or a
protein that binds with a receptor is modified with a fluorine
containing moiety and/or a paramagnetic ion to produce an analog
that is useful in MRI imaging processes as imaging probes,
diagnostic agents and contrast agents.
[0038] In one embodiment an alginate is employed as the
polyuronide. Alginic acid is a linear 1,4-linked block copolymer
comprised of .beta.-D-mannuronic acid (M) and .alpha.-L-guluronic
acid (G) residues. Alginate may come with different
mannuronate/guluronate ratios and compositions. Alginates with high
G content display the strongest gel-forming ability, based on its
diaxial conformation compared to the diequatorial linkage in polyM
sequences. Typical M:G ratios for alginates from different
biological sources may vary from 1.40-1.95 to 0.45-1.00, with high
"G" alginates containing up to 69% G residues compared to
.about.38-41% for regular alginates. The alginate from brown
seaweed Macrocystis pyrifera, for instance has 18% polyG segments,
41% polyM segments and 42% mixed G/M segments, whilst Laminaria
hyperborean alginate has 61% polyG segments, 13% polyM segments and
27% mixed G/M segments. In addition, epimerases may be employed to
alter alginate monosaccharides composition. Preferred alginates for
use in the present invention are high G alginates having at least
about 50% G residues. High G alginates are preferred particularly
in applications requiring a gelled form of the alginate, such as
beads or capsules derived from the well-known complexation with
calcium, barium or similar ions, since they result in higher gel
strength or permit gel-formation at lower alginate concentrations.
These high G alginates are fluorinated as described herein to make
fluorinated high G alginates. When non-gelling alginate
compositions are desired, propylene glycol alginates or high M
alginates can be employed as starting materials as described
herein. Due to their solubility in organic solvents, the propylene
glycol alginates are also preferred when the fluorinations involve
reagents that are very hydrophobic and incompatible with aqueous
solutions. When compositions with no or limited water solubility
are desired, alginic acid itself is a preferred starting material
or the final product can be converted by appropriate ion exchange
processes into the equivalent free alginic acid form. Various other
derivatives and variations on the above methods can be used for
alginate and other polyuronides that are known to those skilled in
the art. Some 200 grades of alginic acid and its various salts are
manufactured and are commercially available.
[0039] In another embodiment Annexin V is modified for the purposes
of the present invention via its N-terminal region in order to
fully retain its membrane binding affinity. Suitable modification
procedures have been described by Tait et al. (see Tait, J. F.,
Gibson, D., Fujikawa, K., J. Bio. Chem., 264, 7944-9, 1989; Tanaka,
K., Einaga, K. Tsuchiyama, H. Tait, J. F. Fujikawa, K.,
Biochemistry., 35, 922-9, 1996; Tait, J. F., Brown, D. S., Gibson,
D., Blankenberg, F. G. Strauss, H. W., Bioconjugate Chem., 11,
918-925, 2000). Analogous procedures can be employed to prepare
other bio-active proteins under suitable conditions to retain
receptor binding activity. The fluorinated Annexin V is then useful
as a diagnostic tool to identify apoptosis in tissues, tumors or
other diseased organs and assess the effectiveness of the
respective therapeutic interventions.
[0040] The paramagnetic compounds of this invention can be used as
contrast-enhancing agents for in vivo MR imaging and magnetic
resonance angiography. The contrast agents are administered orally,
intravascularly or intraperitoneally in physiological buffer or
other physiologically acceptable carriers that are well known to
one of ordinary skill in the art. The dosage depends on the
sensitivity of the NMR imaging instrumentation and on the
composition of the contrast agent. Thus, a contrast agent
containing a highly paramagnetic substance, e.g., gadolinium (III),
generally requires a lower dosage than a contrast agent containing
a paramagnetic substance with a lower magnetic moment, e.g., iron
(III). In general, dosage will be in the range of about 0.001-1
mmol/kg, more preferably about 0.01-0.1 mmol/kg. In one embodiment,
the products are dispersed in a suitable injection medium, such as
distilled water or normal saline, to form a dispersion that is
introduced into the subject's vascular system by intravenous
injection. The particles are then carried through the vascular
system to the target organ where they are taken up.
[0041] When intravascularly administered, the paramagnetic
compounds will be preferentially taken up by organs that ordinarily
function to cleanse the blood of impurities, notably the liver,
spleen, and lymph nodes, and the other organs that tend to
accumulate such impurities, notably bone and neural tissue and to
some extent, lung. In each of these organs and tissues, the uptake
into the reticuloendothelial cells will occur by phagocytosis,
wherein the compounds enter the individual cells in membrane-bound
vesicles; this permits a longer half-life in the cells, as such
membrane-bound paramagnetic compounds will not tend to clump or
aggregate (aggregates are rapidly metabolized and cleared from the
organ/tissue). Other uptake mechanisms are possible, e.g.,
pinocytosis. Also, it is possible that the other cells of the liver
(hepatocytes) may absorb the paramagnetic compounds.
[0042] Because cancerous tumor cells can lack the ability of
phagocytic uptake, the intravascularly administered paramagnetic
compounds can serve as valuable tools in the diagnosis of cancer in
the above-mentioned organs, as tumors will be immediately
distinguishable on any image obtained.
[0043] In another embodiment, the paramagnetic compounds are
administered as dispersions into the gastrointestinal tract that
includes the esophagus, stomach, large and small intestine, either
orally, by intubation, or by enema, in a suitable medium such as
distilled water or any suitable pharmaceutical vehicle. The
particles are preferentially absorbed by the cells of the tract,
especially those of the intestine and, like the intravascularly
introduced particles will exert an effect on T.sub.2 of the organ
or tissue. In this manner, cancers and other debilitating diseases
of the digestive system such as ulcers can be diagnosed and
affected areas pinpointed.
[0044] Preparation of New Imaging Probes
[0045] The novel fluorinated compounds of the present invention
containing a carbohydrate, a polymer or protein backbone or
substrate are obtained by treating the respective starting
materials (backbone or substrate moiety) with fluorine moieties
employing routine fluorination chemistry such as those described
below.
[0046] New fluorinated polymers are prepared as MRI probes. One
target substrate is alginate, which can be fluorinated by a number
of processes. A series of such fluorine-containing, water soluble
alginates can be prepared with a wide range of fluorine contents.
Fluorine contents can vary from 5% to over 40%, and can readily be
further maximized. More importantly, although some line broadening
of the NMR resonances of the fluorine-containing, water soluble
alginates may be observed, their .sup.19F-NMR spectra may display
very high signal to noise (STN) ratios that are of excellent
diagnostic value, even at modest fluorine contents. FIG. 1a shows
the example of a heptafluorobutyryl alginate derivative (Example 1,
F .about.10%) and its .sup.19F-NMR spectrum in dilute aqueous
solution (.about.3 w/v %) acquired with only 100 transients. FIGS.
1a,b shows the six, well-dispersed (.about.45 ppm) trifluoromethyl
and difluoromethylene resonances of this derivative with high
STNs.
[0047] Furthermore, when heptafluorobutyryl alginate was
transformed into beads by addition to aqueous calcium solutions,
the resulting .sup.19F-NMR spectrum (FIG. 1c) was acceptable, even
at the relatively low overall concentrations of this material
(0.4%). Various additional examples of these MRI probes were
prepared (see Examples 2-17), using alginate and other fluorination
approaches. The .sup.19F-NMR spectra of some of the resulting
materials are displayed in FIG. 2. It is evident that in all cases,
the alginate probes produce very suitable NMR spectra with high
STNs, whose .sup.19F-resonances could be tailored to appear in a
broad range of spectral regions, depending on the type of fluorine
residue incorporated. Thus, FIG. 2c shows the .sup.19F-NMR spectrum
of the perfluoroaniline alginate derivative of Example 4, with all
its major resonances appearing between -150 and -175 ppm. The
dominant resonances of the perfluoropolymer alginate derivative of
Example 9 (FIG. 2a) are at -60 to -80 ppm, and those of the
derivative of Example 11 (FIG. 2b) are at -55 to -85 ppm,
respectively. The ability to tailor spectral properties of
polymeric imaging agents by appropriate choice of fluorine
substituents offers clear advantages, particularly when MRI
experiments require resonances in specific spectral regions for
selective pulse sequences. Another benefit is that these new
polymeric imaging agents permit the combined use of standard PFCs
(e.g., in encapsulated form), without causing concerns for
potential spectral overlap of the respective fluorine resonances.
Such multiple .sup.19F probe systems could possibly be designed to
facilitate the simultaneous assessment of several environmental
conditions.
[0048] Linking of fluorinated residues to polyuronides, Annexin V,
and other substrates described herein can be accomplished by a
number of well known reactions, many of which have been described
generally in conjugate chemistry (for reviews see, for instance: G.
T. Hermanson, Bioconjugate Chemistry, Academic Press, New York,
1996; S. S. Wong, Chemistry of protein conjugation and
cross-linking, CRC Press, Boca Raton, 1993; R. L. Lundblad,
Techniques in Protein Modification, CRC Press, Boca Raton, 1994; C.
F. Meares (ed.), Perspectives in Bioconjugate Chemistry, American
Chemical Society, Washington, 1993).
[0049] A terminal hydroxyl group on the polyuronides, proteins
(Annexin V), and other substrates described herein can be allowed
to react with bromoacetyl chloride to form a bromoacetyl ester that
in turn is allowed to react with an amine precursor to form the
--NH--CH.sub.2--C(O)-- linkage. A terminal hydroxyl group also can
be allowed to react with 1,1'-carbonyl-bisimidazole and this
intermediate in turn allowed to react with an amino precursor to
form a --NH--C(O)O-- linkage (see Bartling et al., Nature, 243,
342, 1973). A terminal hydroxyl also can be allowed to react with a
cyclic anhydride such as succinic anhydride to yield a half-ester
which, in turn, is allowed to react with a precursor of the formula
C.sub.xF.sub.yH.sub.z--NH.sub.2 using conventional peptide
condensation techniques such as dicyclohexylcarbodiimide,
diphenylchlorophosphonate, or 2-chloro-4,6-dimethoxy-1,3,5-triazine
(see e.g., Means et al., Chemical Modification of Proteins,
Holden-Day, 1971). A terminal hydroxyl group can also be allowed to
react with 1,4-butanediol diglycidyl ether to form an intermediate
having a terminal epoxide function linked to the polymer through an
ether bond. The terminal epoxide function, in turn, is allowed to
react with an amino or hydroxyl precursor (Pitha et al., Eur. J.
Biochem., 94, 11, 1979; Elling and Kula, Biotech. Appl. Biochem.,
13, 354, 1991; Stark and Holmberg, Biotech. Bioeng., 34, 942,
1989).
[0050] Halogenation of a hydroxyl group permits subsequent reaction
with an alkanediamine such as 1,6-hexanediamine. The resulting
product then is allowed to react with carbon disulfide in the
presence of potassium hydroxide, followed by the addition of
proprionyl chloride to generate a isothiocyanate that in turn is
allowed to react with an amino precursor to yield a
--N--C(S)--N----(CH.sub.2).sub.6--NH-- linkage (see e.g., Means et
al., Chemical Modification of Proteins, Holden-Day, 1971).
[0051] A carboxylic acid group of the polyuronides, proteins
(Annexin V), and other substrates described herein can be activated
with N,N'-dicyclohexylcarbodiimide,
1-(3-dimethylaminopropyl)-3-ethylcarbodiim- ide or equivalent
carbodiimides and then allowed to react with an amino or hydroxyl
group to form an amide or ether respectively. Anhydrides and acid
chlorides will produce the same links with amines and alcohols.
Alcohols can be activated by carbonyldiimidazole and then linked to
amines to produce urethane linkages. Alkyl halides can be converted
to amines or allowed to react with an amine, diamines, alcohols, or
diol. A hydroxy group can be oxidized to form the corresponding
aldehyde or ketone. This aldehyde or ketone then is allowed to
react with a precursor carrying a terminal amino group to form an
imine that, in turn, is reduced with sodium borohydride or sodium
cyanoborohydride to form the secondary amine (see Kabanov et al.,
J. Controlled Release, 22, 141 (1992); Methods Enzymology, XLVII,
Hirs & Timasheff, Eds., Acad. Press, 1977). The precursor
terminating in an amino group can also be allowed to react with an
alkanoic acid or fluorinated alkanoic acid, preferably an activated
derivative thereof, such as an acid chloride or anhydride, to form
a linking group --CONH--. Alternatively, an amino precursor can be
treated with an .alpha.-.omega.-diisocyanoalkane to produce an
--NC(O)NH(CH.sub.2).sub.6NHC(O)--N-- linkage (see Means, Chemical
Modification of Proteins, Holden-Day, 1971). Furthermore, linkages
that are unsymmetrical, such as --CONH-- or --NHCOO--, can be
present in the reverse orientation; e.g., --NHCO-- and --OCONH--,
respectively. Examples of an activated carbonyl group include
anhydride, ketone, p-nitrophenylester, N-hydroxysuccinimide ester,
pentafluorophenyl ester and acid chloride.
[0052] Suitable fluorinated starting materials for making the novel
compositions of the present invention include both organic and
inorganic fluorinating agents. Representative fluorinating agents
include trifluoromethylhypofluorite, sulfur tetrafluoride,
CF.sub.2Cl, FSO.sub.2[CF.sub.2].sub.xCF.sub.3 (where x=1-20),
potassium fluoride, organic fluorinating agents, such as
fluoroamine, fluorocarbamate, fluorotriazine, fluorosulfonylalkyl
derivatives, Selectfluor.TM., fluoroalkylcarboxylic acids,
fluoroalkylaldehydes, anhydrides, esters, ketones, acid chlorides
of fluoroalkylcarboxylic acids, such as monofluoroacetic acid,
difluoroacetic acid, trifluoroacetic acid, pentafluoro-propionic
acid, heptafluorobutyric acid, heptafluorobutyric anhydride,
heptafluorobutyrylchloride, nonafluoropentanoic acid,
tridecafluoroheptanoic acid, pentadecafluorooctanoic acid,
heptadecafluorononanoic acid, nonadecafluorodecanoic acid,
perfluorododecanoic acid, perfluorotetradecanoic acid;
fluoroalkanols, such as 2,2,3,3,4,4,4-heptafluoro-1-butanol,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,- 8-penta-decafluoro-1-octanol,
2,2,3,3,4,4,5,5,6,6,7,7,8,9,9,9-hepta-decafl- uoro-1-nonanol,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadeca-fluoro-1-
-decanol, Krytox and Zonyl derivatives, fluoroarylesters,
fluoroalkylamines, fluoroarylamines,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,1-
0,11,11,11-heneicosafluoro-1-undecanol; fluorinated polymers
containing reactive terminal groups, fluoroalkyl halides, such as
perfluoroethyl iodide, perfluoropropyl iodide, perfluorohexyl
bromide, perfluoroheptyl bromide, perfluorooctyl bromide,
perfluorodecyl iodide, perfluorooctyl iodide,
1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8-heptadecafluoro-10-iododecane,
1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8-heptadecafluoro-10-iododecane,
polytetrafluoro-ethyleneoxide-co-difluoromethyleneoxide-.alpha.,.omega.-b-
is(methylcarboxylate), dihydroxypropanoxymethyl derivatives of
perfluoropolyoxyalkane, hydroxypolyethylenoxy derivatives of
perfluoropolyoxyalkane and the like. Suitable modification
procedures have been described in several monographs (J. J. Clark,
D. Walls. T. W. Bastock, Aromatic Fluorination, CRC Press, Boca
Raton, Fla., 1996; M. Hudlicky, A. E. Pavlath, Chemistry of Organic
Fluorine Compounds, ACS, Washington, D.C. 1995; M. Howe-Grant ed.,
Fluorine Chemistry, A Comprehensive Treatment, Wiley, New York,
1995; G. A. Olah, G. K. Sarya Prakash, R. D. Chambers, eds.
Synthetic Fluorine Chemistry, Wiley, New York, 1992).
[0053] Specific examples of compounds of formulas I-IV may require
the use of protecting or blocking groups to enable their successful
elaboration into the desired structure. Protecting groups may be
chosen with reference to Greene, T. W., et al., Protective Groups
in Organic Synthesis, John Wiley & Sons, Inc., 1991. The
blocking groups are readily removable, i.e., they can be removed,
if needed, by procedures that will not cause cleavage or other
disruption of the remaining portions of the molecule. Such
procedures include chemical and enzymatic hydrolysis, treatment
with chemical reducing or oxidizing agents under mild conditions,
treatment with fluoride ion, treatment with a transition metal
catalyst and a nucleophile, and catalytic hydrogenation.
[0054] Examples of suitable hydroxyl protecting groups are:
trimethylsilyl, triethylsilyl, o-nitrobenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl, t-butyldiphenylsilyl,
t-butyldimethylsilyl, benzyloxycarbonyl, t-butyloxycarbonyl,
2,2,2-trichloroethyloxycarbonyl, and allyloxycarbonyl. Examples of
suitable carboxyl protecting groups are benzhydryl, o-nitrobenzyl,
p-nitrobenzyl, 2-naphthylmethyl, allyl, 2-chloroallyl, benzyl,
2,2,2-trichloroethyl, trimethylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, 2-(trimethylsilyl)ethyl, phenacyl,
p-methoxybenzyl, acetonyl, p-methoxyphenyl, 4-pyridylmethyl and
t-butyl.
[0055] Paramagnetic polyuronides can be prepared by contacting an
appropriate salt forming polyuronide with an equimolar amount of an
appropriate metal ion under conditions sufficient to form a
polyuronide salt. For example, paramagnetic alginate beads can be
formed by slowly adding sodium alginate having a high guluronic
acid content (>50%) to a stirred aqueous solution of gadolinium
(III) acetate at about an equal molar ratio, or at an appropriate
different ratio, depending on the end use. The resulting beads that
are formed are isolated by centrifugation and can be washed with
calcium chloride to remove excess gadolinium. Similarly,
superparamagnetic polyuronide beads can be prepared employing the
same procedures described above but substituting a suspension of
superparamagnetic nanoparticles, such as, iron oxide particles, for
the aqueous gadolinium.
[0056] Superparamagnetic protein conjugates are prepared by mixing
superparamagnetic nanoparticles with a buffered protein solution.
The superparamagnetic nanoparticles can optionally be oxidized
prior to the reaction with the protein. The reaction mixture is
then centrifuged and the supernatant is discarded to give the
desired protein conjugate.
[0057] The compounds of the present invention can be prepared
readily according in the following detailed examples using readily
available starting materials, reagents and conventional synthetic
procedures. Additional variants are also possible that are known to
those of ordinary skill in this art, but that are not mentioned in
greater detail. The following examples illustrate the practice of
the present invention but should not be construed as limiting its
scope.
MATERIALS
[0058] Alginic acid, sodium alginate, high G alginate,
propyleneglycol alginate 3,5,5'-tris(trifluoromethyl)
octafluorohexanoic acid, 3,5,5'-tris(trifluoromethyl)
octafluorohexanoic acid, 3,5,5'-tris(trifluoromethyl)
octafluorohexanol, perfluoro-3,6,9-trioxatri- decanoic acid methyl
ester, methyl perfluorohexadecanoate, dextran, poly(ethylene
glycol), and superparamagnetic iron oxide nanoparticles (3 nm) were
obtained from CarboMer, Inc., Westborough, Mass. and San Diego,
Calif.; the alginic acid, sodium alginate, high G alginate,
propyleneglycol alginate starting materials had molecular weights
of .about.600,000, .about.500,000, .about.450,000, and
.about.700,000 Da, respectively. The hexafluoropropane oxide and
heptafluorobutyryl chloride were obtained from Lancaster Synthesis,
Windham, N.H. Deoxo-Fluor [bis(2-methoxyethyl)aminosulfur trioxide]
was obtained from Air Products, Allentown, Pa.
Polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.al-
pha.,.omega.-bisdifluoroacetic acid, gadolinium (III) acetate,
polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.,.omega.-bi-
s(methylcarboxylate),
polytetrafluoroethyleneoxide-co-difluoromethyleneoxi-
de-.alpha.,.omega.-diisocyanate and Annexin V were obtained from
Aldrich, St. Louis, Mo.
[0059] The formulas in the following examples and claims include m
and n subscripts which are included to designate the ratio of
repeating units. In each formula the sum of m and n is one (1), ie
m+n=1.
EXAMPLE 1
[0060] Heptafluorobutyryl Alginic Acid
[0061] A solution of heptafluorobutyryl chloride in DMSO (0.6
equivalents) was added to alginic acid and stirred at ambient
temperature for 6 hours. The product was precipitated with acetone,
filtered, washed with acetone, dialyzed and dried, yielding
heptafluorobutyryl alginic acid with F 9.07%. 2
[0062] wherein m+n=1
[0063] Biocompatibility results heptafluorobutyryl alginate
(Example 1) was tested in human cell culture and found to be
non-toxic at concentrations of 0.2-1.0%.
EXAMPLE 2
[0064] 6-[3-[2-(Perfluorohexyl)-2-ethoxy]-2-hydroxypropyl] Alginic
Acid
[0065] A solution of
3-[2-(perfluorohexyl)-2-ethoxy]-1,2-epoxypropane in methylene
chloride (0.6 equivalents) was added to alginic acid and stirred at
ambient temperature for 6 hours.
[0066] The suspension was filtered, washed with methylene chloride
and acetone, dialyzed and dried, yielding
3-[2-(perfluorohexyl)-2-hydroxy] alginate with F 20.41%. 3
[0067] wherein m+n=1
EXAMPLE 3
[0068] Perfluoro tri-n-butylamine Alginate
[0069] A solution of perfluoro tri-n-butylamine in water (1.1
equivalents) was added to alginic acid and stirred at ambient
temperature for 6 hours. The product was precipitated with acetone,
filtered, washed with acetone, dialyzed and dried, yielding
perfluoro tri-n-butylamine alginate with F 9.41% 4
[0070] wherein m+n=1
EXAMPLE 4
[0071]
Poltetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.-methy-
l-carboxylate-.omega.-carboxylate Propylene Glycol Alginate
[0072] A solution of propylene glycol alginate in methanol was
treated with
polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.,.omeg-
a.-bis(methylcarboxylate) (Mw .about.2,000 Da, 0.6 equivalents) and
the resulting viscous paste was stirred at ambient temperature for
6 hours. The reaction mixture was precipitated with acetone, washed
with acetone, filtered, dialyzed and dried, yielding the
perfluoropolymer-labeled alginate with F 27.66%. 5
[0073] wherein m+n=1
EXAMPLE 5
[0074] 6-[2-(Perfluorohexyl)-2-hydroxy] DANSYL Alginic Acid
[0075] A solution of 6-[2-(perfluorohexyl)-2-hydroxy] alginate
(Example 2, 0.6 equivalents) in DMSO was treated with
5-N,N-dimethylamino-1-naphthale- nsulfonyl (DANSYL) chloride (0.1
equivalents) dissolved in dry acetone and sodium carbonate (0.1
equivalents) and stirred at ambient temperature for 3 hours. The
reaction mixture was precipitated with acetone, washed with
acetone, filtered, dialyzed and dried, yielding Dansylated
6-[2-(perfluorohexyl)-2-hydroxy] alginate. 6
[0076] wherein m+n=1
EXAMPLE 6
[0077] 3-[(Hexafluoropropyl)-2-hydroxyl Alginic Acid
[0078] A solution of hexafluoropropyleneoxide in methylene chloride
(0.8 equivalents) was added to sodium alginate with high guluronic
acid content (68%) and stirred at ambient temperature for 6 hours.
The suspension was filtered, washed with methylene chloride and
acetone, dialyzed and dried, yielding
3-[(hexafluoropropyl)-2-hydroxy] alginate with F 12.50%. 7
[0079] wherein m+n=1
EXAMPLE 7
[0080] Perfluorophenylhydrazone Alginic Acid
[0081] A solution of perfluorophenylhydrazine in methylene chloride
(0.6 equivalents) was added to an aqueous solution of sodium
alginate with high guluronic acid content (68%) and stirred at
ambient temperature for 6 hours. The reaction mixture was
precipitated with acetone, washed with acetone, filtered, dialyzed
and dried, yielding perfluorophenylhydrazone alginate with F
10.35%. 8
[0082] wherein m+n=1
EXAMPLE 8
[0083]
Polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.-difl-
uoroacetic acid-.omega.-difluoroacetyl Fluorescein Alginate
[0084] An aqueous solution of sodium alginate with high guluronic
acid content (68%) was treated with
polytetrafluoroethyleneoxide-co-difluorome-
thyleneoxide-.alpha.,.omega.-bisdifluoroacetic acid (Mw .about.500,
0.6 equivalents) and the resulting viscous paste was stirred at
ambient temperature for 6 hours. A portion was treated with
fluorescein isothiocyanate (0.1 equivalents) for 4 hours. The
reaction mixture was precipitated with acetone, washed with
acetone, filtered, dialyzed and dried, yielding perfluoropolymer-
and FITC-labeled alginate with F 22.64%. 9
[0085] wherein m+n=1
EXAMPLE 9
[0086]
Polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.-difl-
uoroacetic acid-.omega.-difluoroacetyl Propylene Glycol
Alginate
[0087] A solution of propylene glycol alginate in methanol was
treated with
polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.,.omeg-
a.-bisdifluoroacetic acid (Mw .about.2,000, 0.6 equivalents) and
the resulting viscous paste was stirred at ambient temperature for
6 hours. The reaction mixture was precipitated with acetone, washed
with acetone, filtered, dialyzed and dried, yielding
perfluoropolymer-labeled alginate with F 33.04%. 10
[0088] wherein m+n=1
EXAMPLE 10
[0089] Perfluorobenzamide Alginate
[0090] A solution of perfluoroaniline (0.6 equivalents) in
methylene chloride was added to an aqueous solution of sodium
alginate with high guluronic acid content (68%) and stirred at
ambient temperature for 6 hours. The reaction mixture was
precipitated with acetone, washed with acetone, filtered, dialyzed
and dried, yielding perfluorobenzamide alginate with F 19.35%.
11
[0091] wherein m+n=1
EXAMPLE 11
[0092] Perfluoroaniline Alginate
[0093] A solution of perfluoroaniline (0.6 equivalents) in
methylene chloride was added to an aqueous solution of aqueous
sodium alginate with high guluronic acid content (68%). Sodium
cyanoborohydride (8.6 equivalents) was added and stirred at ambient
temperature for 6 hours. The reaction mixture was precipitated with
acetone, washed with acetone, filtered, dialyzed and dried,
yielding perfluoroaniline alginate with F 22.65%. 12
[0094] wherein m+n=1
EXAMPLE 12
[0095] 3-[(Hexafluoropropyl)-2-hydroxy] Propyleneglycol
Alginate
[0096] A solution of hexafluoropropyleneoxide in methylene chloride
(0.7 equivalents) was added to propyleneglycol alginate and stirred
at ambient temperature for 6 hours. The suspension was filtered,
washed with methylene chloride and acetone, dialyzed and dried,
yielding 3-[(hexafluoropropyl)-2-hydroxy]propyleneglycol alginate
with F 20.41%. 13
[0097] wherein m+n=1
EXAMPLE 13
[0098]
Polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.-toly-
lurethane-.omega.-tolylisocyanate Propylene Glycol Alginate
[0099] A solution of propylene glycol alginate in methanol was
treated with
polytetrafluoroethyleneoxide-co-difluoromethyleneoxide-.alpha.,.omeg-
a.-diisocyanate (Mw .about.3,000, 0.6 equivalents) and the
resulting viscous paste was stirred at ambient temperature for 6
hours. The reaction mixture was precipitated with acetone, washed
with acetone, filtered, dialyzed and dried, yielding
perfluoropolymer-labeled alginate with F 29.99%. 14
[0100] wherein m+n=1
EXAMPLE 14
[0101] Perfluoro-3,6,9-trioxatridecanoate Alginate
[0102] An aqueous solution of alginate with high guluronic acid
content (68%) was treated with perfluoro-3,6,9-trioxatridecanoic
acid methyl ester (1.6 equivalents) and the resulting viscous paste
was stirred at ambient temperature for 16 hours. The reaction
mixture was precipitated with acetone, washed twice with acetone,
filtered, dialyzed and dried, yielding
perfluoro-3,6,9-trioxatridecanoate alginate with F 30.25%. 15
[0103] wherein m+n=1
EXAMPLE 15
[0104] 3,5,5'-Tris(trifluoromethyl)octafluorohexanoate Alginate
[0105] A dispersion of alginate with high guluronic acid content
(68%) in acetone was treated with 1,3-diisopropylcarbodiimide (0.8
equivalents) for 1 hour and then with 3,5,5'-tris(trifluoromethyl)
octafluorohexanol (0.6 equivalents) and the resulting viscous Paste
was stirred at ambient temperature for 16 hours. The reaction
mixture was precipitated with acetone, washed twice with acetone,
filtered, dialyzed and dried, yielding the
perfluoro-3,5,5'-trimethylhexanoate alginate with F 24.73%. 16
[0106] wherein m+n=1
EXAMPLE 16
[0107] 3,5,5'-Tris(trifluoromethyl)octafluorohexanoate Propylene
Glycol Alginate
[0108] A solution of propylene glycol alginate in methanol was
treated with 3,5,5'-tris(trifluoromethyl)octafluorohexanoic acid
(0.4 equivalents) and the resulting viscous paste was stirred at
ambient temperature for 16 hours. The reaction mixture was
precipitated with acetone, washed twice with acetone, filtered,
dialyzed and dried, yielding the
perfluoro-3,5,5'-trimethylhexanoate-labeled alginate with F 14.71%.
17
[0109] wherein m+n=1
EXAMPLE 17
[0110] Methyl Perfluorohexadecanoate Alginate
[0111] An aqueous solution of alginate with high guluronic acid
content (68%) was treated with methyl perfluorohexadecanoate (0.4
equivalents) and the resulting viscous paste was stirred at ambient
temperature for 16 hours. The reaction mixture was precipitated
with acetone, washed twice with acetone, filtered, dialyzed and
dried, yielding the methyl perfluorohexadecanoate alginate with F
32.36%. 18
[0112] wherein m+n=1
EXAMPLE 18
[0113] Paramagnetic Gadolinium Alginate Beads
[0114] To a rapidly stirred, aqueous solution of gadolinium (III)
acetate (1.1 equivalents) was added dropwise a dilute aqueous
solution of sodium alginate with high guluronic acid content (68%)
through a syringe. The resulting gel beads were centrifuged, the
supernatant was discarded, and the beads were washed and
centrifuged twice with dilute calcium chloride solution to remove
excess gadolinium. A magnetization curve revealed that the alginate
beads were paramagnetic. Sterile preparations were obtained, by
filter sterilizing the component solutions through 0.22 micron
filters prior to their combination. Paramagnetic gadolinium
alginate beads (Example 18) were found to be non-toxic in cell
culture.
EXAMPLE 19
[0115] Superparamagnetic Iron Oxide Alginate Beads
[0116] To a rapidly stirred suspension of superparamagnetic iron
oxide nanoparticles (3 nm, 1.1 equivalents) in a 10 mmol aqueous
calcium chloride solution containing poly(ethylene glycol) (NF
grade, Mw 400, 25%) was added dropwise with a syringe to a dilute
aqueous solution of sodium alginate with high guluronic acid
content (68%). The resulting gel beads were centrifuged, the
supernatant was discarded, and the beads were washed and
centrifuged twice with dilute calcium chloride solution. A
magnetization curve revealed that the alginate beads were
superparamagnetic. Sterile preparations were obtained by filter
sterilizing the component solution and suspension through 0.22
micron filters prior to their combination.
EXAMPLE 20
[0117] Superparamagnetic Iron Oxide Alginate-Coated
Nanoparticles
[0118] To a rapidly stirred, 10 mmol aqueous calcium chloride
solution was added dropwise through a syringe a suspension of
superparamagnetic iron oxide nanoparticles (3 nm) in a dilute
aqueous solution of sodium alginate with high guluronic acid
content (68%). The resulting coated nanoparticles were centrifuged,
the supernatant was discarded, and the nanoparticles were washed
and centrifuged twice with dilute calcium chloride solution.
EXAMPLE 21
[0119] Superparamagnetic Iron Oxide Alginate-Coated
Nanoparticles
[0120] To a rapidly stirred, dilute aqueous solution of sodium
alginate with high guluronic acid content (68%) was slowly added a
suspension of superparamagnetic iron oxide nanoparticles (3 nm) in
a poly(ethylene glycol) (NF grade, Mw 400, 25%). The resulting
suspension was stirred for 10 minutes, then heated for 30 minutes
at 70.degree. C. and centrifuged. The supernatant was discarded,
and the coated nanoparticles were dialyzed.
EXAMPLE 22
[0121] Superparamagnetic Iron Oxide Dextran-Coated
Nanoparticles
[0122] To a rapidly stirred, dilute aqueous dextran (Mw 10,000)
solution was slowly added a suspension of superparamagnetic iron
oxide nanoparticles (3 nm) in a poly(ethylene glycol) (NF grade, Mw
400, 25%). The resulting suspension was stirred for 10 minutes,
then heated for 30 minutes at 70.degree. C. and centrifuged The
supernatant was discarded, and the coated nanoparticles were
dialyzed.
EXAMPLE 23
[0123] Superparamagnetic Iron Oxide Annexin Conjugate
[0124] To a rapidly stirred, aqueous solution of superparamagnetic
iron oxide dextran-coated nanoparticles from Example 22 was added
sodium paraperiodate (0.05 equivalents) and the resulting mixture
was stirred for 1 h. Ethylene glycol was added and the mixture was
then centrifuged. The supernatant was discarded, and the oxidized,
coated nanoparticles were washed and centrifuged twice with PBS
buffer. The oxidized, coated nanoparticles were added to a buffered
solution of Annexin V. The resulting mixture was stirred for 25
minutes at 4.degree. C. and then centrifuged. The supernatant was
discarded, to give the desired conjugate.
EXAMPLE 24
[0125] Superparamagnetic Iron Oxide Annexin Conjugate
[0126] Following the procedure outlined for Example 23,
alginate-coated nanoparticles prepared according to Examples 20 or
21 were used either with the oxidation step, or directly, to give
the desired conjugates.
EXAMPLE 25
[0127] Fluorescent, Superparamagnetic Iron Oxide Annexin
Conjugate
[0128] Following the procedure outlined for Examples 22 and 23,
fluorescein isocyanate labeled Annexin V was used instead of the
unlabeled protein to give the desired fluorescent conjugates with
coated superparamagnetic iron oxide nanoparticles. Alternatively,
fluorescent alginate (e.g., Examples 5 and 8) could be
employed.
EXAMPLE 26
[0129] Heptafluorobutyryl Hydroxyethyl Starch
[0130] A solution of heptafluorobutyryl chloride in DMSO (0.6
equivalents) was added to hydroxyethyl starch and stirred at
ambient temperature for 12 hours. The product was precipitated with
acetone, filtered, washed with acetone, dialyzed and dried,
yielding heptafluorobutyryl hydroxyethyl starch with F 16.75%.
EXAMPLE 27
[0131] Perfluorotri-n-butylamine Alginate
[0132] An aqueous acidic dispersion of alginic acid was treated
with perfluorotri-n-butylamine (1.6 equivalents) at ambient
temperature for 12 hours. The product was precipitated with
alcohol, filtered, washed with acetone, dialyzed and dried,
yielding perfluorotri-n-butylamine alginate with F 11.50%.
[0133] Uses of Novel Imaging Agents
[0134] The fluorinated polyuronides and in particular the
fluorinated alginates of the present invention display sensitivity
in their T.sub.1. relaxation times to different oxygen partial
pressures (pO.sub.2), producing linear correlation over a range of
pO.sup.2. This demonstrates their utility as oxygen sensitive
imaging probes. The fluorinated alginates also display chemical
shift and temperature sensitivity, indicating their utility as
temperature sensitive imaging probes. These novel agents of this
invention are suitable for many diagnostic uses, and provide the
ability to image in vivo or non-invasively monitor tissues, organs
and cellular implants, for example, pancreatic islet .beta.-cells
that are encapsulated with the present fluorinated polyuronides,
and measure their mass, function, viability or evidence of
inflammation. Additionally, engraftment of transplanted isolated
pancreatic islets can be monitored, using, for example, islets
labeled with .beta.-cell specific oxygen-sensitive fluorinated
probes. .sup.19F-MRI with these novel agents permits monitoring of
other disorders, such as cancer, the comparison of normal or
diseased cells, organs or tissues, the viability of transplanted
cells or other tissues when those fluorinated agents have
specificity for target tissues. This new methodology is
instrumental in the development of clinical examinations for
monitoring disease progress and response to therapy in diabetics
and in people strongly at risk for diabetes and other patient
populations.
[0135] The simultaneous incorporation of .sup.19F or
superparamagnetic residues and fluorescent moieties into the
polyuronides or polymeric agents affords diagnostic probes that can
be employed for both MRI and fluorescent studies. Examples of such
dual function diagnostic probes are those polyuronides or proteins
that contain both a fluorine moiety as described herein and a
fluorescent moiety or a fluorinated fluorescent moiety such as:
4-trifluoromethyl-7-aminocoumarin, 4-trifluoromethyl-umbelliferone
(or its acetate or butyrate derivatives),
4-fluoro-7-sulfamyl-benzofurazam, certain BODIPY dyes, e.g.,
N-(4,4'-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)-methyli-
odoacetamide,
N-(4,4'-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-in-
dacene-2-yl)-iodoacetamide and
4,4'-difluoro-5-phenyl-4-bora-3a,4a-diaza-s- -indacene-3-propionic
acid, 3-chloro-1-(3-chloro-5-(trifluoromethyl)-2-pyr-
idimyl)-5-(trifluoromethyl)-2[1H]-pyridinone,
6-carboxymethylthio-2',4,'5,-
7'-tetrabromo-4,5,7-trifluorofluorescein (Eosin F3S), and Oregon
Green carboxylic acid.
[0136] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
REFERENCES
[0137] 1. U.S. Pat. No. 6,019,959 Feb. 1, 2000 Oligomeric compounds
that contain perfluoroalkyl, process for their production, and
their use in NMR diagnosis
[0138] INVENTOR(S)--Platzek, Johannes; Niedballa, Ulrich; Raduchel,
Bernd; Schlecker, Wolfgang; Weinmann, Hanns-Joachim; Frenzel,
Thomas; Misselwitz, Bernd; Ebert, Wolfgang
[0139] PATENT ASSIGNEE(S)--Schering Aktiengesellschaft
[0140] 2. U.S. Pat. No. 6,011,048 dated Jan. 4, 2000 Thiazole
benzenesulfonamides as .beta.3 agonists for treatment of diabetes
and obesity
[0141] INVENTOR(S)--Mathvink; Robert J.; Parmee; Emma R.; Tolman;
Samuel; Weber; Ann E.
[0142] PATENT ASSIGNEE(S)--Merck & Co., Inc.
[0143] 3. U.S. Pat. No. 5,510,496 dated Apr. 23, 1996 Substituted
pyrazolyl benzenesulfonamides
[0144] INVENTOR(S)--Talley; John J.; Penning; Thomas D.; Collins;
Paul W.; Malecha; James W.; Bertenshaw; Stephen R.; Graneto;
Matthew J.
[0145] PATENT ASSIGNEE(S)--G. D. Searle & Co.
[0146] 4. U.S. Pat. No. 5,342,823 dated Aug. 30, 1994
Sulfonylureas
[0147] INVENTOR(S)--Kuhlmeyer; Rainer; Topfl; Werner; Fory;
Werner
[0148] PATENT ASSIGNEE(S)--Ciba-Geigy Corporation
[0149] 5. U.S. Pat. No. 6,218,464 dated Apr. 17, 2001 Preparation
of fluorinated polymers
[0150] INVENTOR(S)--Parker; Hsing-Yeh; Lau; Willie; Rosenlind; Erik
S.
[0151] PATENT ASSIGNEE(S)--Rohm and Haas Company
[0152] 6. U.S. Pat. No. 5,798,406 dated Aug. 25, 1998 Fluorinated
acrylic and methacrylic latices and mixtures thereof, processes for
manufacturing them and their applications in the field of
hydrophobic coatings
[0153] INVENTOR(S)--Feret; Bruno; Sarrazin; Laure; Vanhoye;
Didier
[0154] PATENT ASSIGNEE(S)--Elf Atochem S. A
[0155] 7. PCT WO 00/40252, dated 2000 Hetero-polysaccharide
conjugate and methods of making and using the same
[0156] INVENTOR(S)--C. Fraker, L. Invaeradi, M. Mares-Guia, C.
Ricordi
[0157] PATENT ASSIGNEE(S)--Biomm, Inc. & University of
Miami
* * * * *